Kaohsiung J Med Sci July 2007 • Vol 23 • No 7
332
Chronic hepatitis C (CHC) infection is a major health problem worldwide. The global prevalence is esti-mated to be 3%, ranging from 0.1% to 12% in differ-ent countries [1,2]. It is estimated that there are 170 million hepatitis C virus (HCV) carriers in the world. The incidence of new symptomatic infections has been Received: September 19, 2006 Accepted: November 20, 2006
Address correspondence and reprint requests to: Dr Jee-Fu Huang, Division of Hepatobiliary, Department of Internal Medicine, Kaohsiung Medical University Hospital, 100 Tzyou 1stRoad, Kaohsiung 807, Taiwan.
E-mail: jeefuhuang@yahoo.com.tw
Q
UALITATIVE
A
PPLICATION OF
COBAS
AMPLICOR HCV T
EST
V
ERSION
2.0 A
SSAYS IN
P
ATIENTS WITH
C
HRONIC
H
EPATITIS
C V
IRUS
I
NFECTION AND
C
OMPARISON OF
C
LINICAL
P
ERFORMANCE WITH
V
ERSION
1.0
Ming-Yen Hsieh,1,2Li-Po Lee,1Nai-Jen Hou,1,3Jeng-Fu Yang,1,4Jee-Fu Huang,2,3Chia-Yen Dai,2,3,5,6 Wan-Long Chuang,1,6Zu-Yau Lin,1,6Shinn-Cherng Chen,1,6Ming-Yuh Hsieh,1,6
Liang-Yen Wang,1,6Wen-Yu Chang,1,6and Ming-Lung Yu1,4,6
1Division of Hepatobiliary Medicine, Department of Internal Medicine, and 4Department of Preventive
Medicine, Kaohsiung Medical University Hospital, Departments of 3Internal Medicine and
5Occupational and Environmental Medicine, Kaohsiung Municipal Hsiao-Kang Hospital,
2Graduate Institute of Medicine, and 6Faculty of Medicine, College of Medicine,
Kaohsiung Medical University, Kaohsiung, Taiwan.
The objective of this research was to investigate the clinical performance of COBAS AMPLICOR hepatitis C virus (HCV) test version 2.0 Assays (CA V2.0). Eight serial samples with standard HCV ribonucleic acid (RNA) concentration and 10 times serial dilution of the 500 IU/mL samples were tested in triplicate by CA V2.0 (the limit of detection was 50 IU/mL). HCV RNA was inves-tigated with CA V2.0 in 220 specimens from 100 chronic hepatitis C (CHC) patients, 60 chronic hepatitis B patients, and 60 healthy blood donors. The sensitivity was 99% and the specificity was 98.3%. Sera of 84 naïve CHC patients receiving standard interferon plus ribavirin for 24 weeks were tested by CA V2.0 and CA V1.0 at weeks 2, 4 and 8. The positive detection rates of CA V2.0
were significantly higher than CA V1.0 at week 2 (60.7% vs. 51.2%; p< 0.01) and week 8 (27.4% vs.
21.4%; p< 0.05). At weeks 2, 4 and 8, the positive predictive values were 90.91%, 83.02% and
78.69% with CA V2.0, and 90.24%, 82.14% and 72.73% with CA V1.0. The negative predictive val-ues were 58.82%, 77.42% and 86.96% with CA V2.0, and 67.44%, 82.14% and 83.33% with CA V1.0. However, there was no significant difference between CA V2.0 and CA V1.0 for predicting sustained virologic response.
Key Words:hepatitis C virus, hepatitis C virus RNA, interferon therapy, qualitative hepatitis C virus RNA assay
estimated to be 1–3 cases per 100,000 persons annually [1]. Because the majority of cases are asymptomatic, the actual incidence of new infections is obviously much higher.
The enzyme immunoassays (EIAs) that detect anti-bodies to HCV cannot differentiate between active and resolved infection. Qualitative HCV RNA assays detect viral genomes and therefore can both confirm the presence of active infection and demonstrate its presence 4–6 weeks before antibody seroconversion takes place [3]. The application of polymerase chain reaction (PCR) methods for identification of the HCV RNA thus provides important data relating to diag-nosis, monitoring, and treatment of HCV infection. Moreover, recent studies have shown that treatment strategies were mainly determined or tailored by HCV RNA measurements before and/or during treatment [4,5]. Therefore, qualitative HCV RNA assays have become an essential tool for both the diagnosis and monitoring of HCV infection.
HCV RNA can usually be detected in the patient’s serum within 10–14 days after infection [3]. In clinical practice, qualitative HCV RNA assay offers the desired sensitivity and specificity for detecting and confirming the presence of active infection and for documenting the response after antiviral treatment. The detection of HCV RNA by reverse transcription–polymerase chain reaction (RT–PCR) has been widely developed with standard ready-to-use assays such as the AMPLICOR HCV test. This test was successfully automated by using an integrated PCR system based on COBAS AMPLICOR technology that fully automates all steps of PCR amplification and detection. Developed and manufactured by Roche Diagnostics System, the COBAS AMPLICOR HCV RNA assay version 1.0 (CA V1.0; Roche Diagnostic Systems, Branchburg, NJ, USA) is available worldwide. Recently, a new version (version 2.0) of the COBAS AMPLICOR HCV RNA assay (CA V2.0; Roche Diagnostic Systems) has been developed and marketed.
In this study, we aimed to evaluate the clini-cal performance of these two versions of COBAS AMPLICOR HCV RNA assays in terms of sensitivity, specificity, and reproducibility. We also attempted to compare the performance characteristics for the pre-diction of sustained virologic response (SVR) between these two versions among CHC patients receiving standard interferon (IFN) and ribavirin combination therapy.
M
ATERIALS ANDM
ETHODSValidation test and detection limit test
For reliability test, eight validation nucleic acid panels with standard HCV RNA concentration (NAP-HCV 000; 0 IU/mL, and NAP-HCV 001-007; 50, 500, 5,000, 50,000, 200,000, 500,000, and 2,000,000 IU/mL; Acro-Metrix, USA) were processed in triplicate with CA V2.0 (24 tests were performed).To confirm the limit of detection, NAP-HCV 002 (500 IU/mL) was used for serial dilution in ratios of 1:10; 1:100; 1:1,000; and 1:10,000 (from 50 IU/mL to 0.05 IU/mL). All diluted specimens were tested in trip-licate three times (total nine tests in each concentration) with CA V2.0 assay.
Sensitivity and specificity tests
The presence of HCV RNA was investigated with CA V2.0 in 220 specimens consecutively collected from 100 CHC patients, 60 chronic hepatitis B (CHB) patients, and 60 healthy blood donors. Serum samples from enrolled individuals were collected on SST Vacutainer (Becton-Dickinson, Meylan, France), centrifuged,
ali-quoted, stored at −30°C within 90 minute of collection,
and tested with the assay within a period of 3 months. Eligibility criteria for CHC patients were defined as: anti-HCV (EIA 3.0; Abbott, North Chicago, IL, USA) positivity; consecutive serum alanine aminotransferase levels more than 1.5 times above the upper normal limit for more than 6 months; chronic hepatitis proven by liver histopathology; negative for HBsAg (enzyme-linked immunosorbent assay [ELISA]; Abbott); and patients without concomitant potential causes of chronic hepatitis other than HCV infection, such as alcoholism, drug-induced scenarios, autoimmune hepatitis, primary biliary cirrhosis, Wilson’s disease, α-antitrypsin deficiency, etc. Healthy blood donors were defined as follows: (1) negative for anti-HCV (EIA 3.0) and HBsAg (ELISA); (2) normal liver func-tion test; and (3) no history of drug abuse, transfu-sion, hepatitis, and of percutaneous risks such as tattooing, dental procedures, or any operation within 1 year. The sensitivity and specificity of CA V2.0 assay was determined directly from the comparison of the results with the biological standards.
Clinical performance
Sera of 84 naïve CHC patients receiving combination therapy with high dose conventional IFN (6 MU
subcutaneously three times weekly) plus oral ribavirin (1,000–1,200 mg daily) for 24 weeks were analyzed for HCV RNA retrospectively. SVR was defined as clearance of serum HCV RNA at the end of treatment and throughout the 24-week follow-up period.
Blood samples were collected at weeks 2, 4 and 8 after initiation of combination therapy. Each sample was processed in parallel for HCV RNA with both CA V2.0 and CA V1.0.
Undetectable HCV RNA was used as a prediction of SVR in CHC patients. In our study, the prognostic values for combination therapy in these 84 patients were analyzed in parallel with both CA V2.0 assay and CA V1.0 assay at weeks 2, 4 and 8. Negative HCV RNA test results were used as a positive predic-tion of SVR, and positive RNA test results indicated a negative prediction of SVR.
Statistical analyses
The discrepancy between these measures for CA
V2.0 and CA V1.0 were analyzed with χ2 tests for
non-independent sample proportions (McNemar’s
test). A p value < 0.05 was considered statistically
significant.
R
ESULTSValidation tests and detection limit tests
A total of 24 tests were performed with CA V2.0 for the validation test (triplicate for eight panel members with standard HCV RNA concentration; NAP-HCV 000-007). Only NAP-HCV 000 (0 IU/mL) showed negative results in the triplicate test and all theother panel members showed positive results with CA V2.0.
For the limit of detection, each serially diluted sample was tested nine times with CA V2.0. Samples with HCV RNA concentration of 50 IU/mL were all positive. However, some negative results were obtained below 50 IU/mL (the detection rate of serial dilutions: 50 IU/mL, 100%; 5 IU/mL, 33.3%; 0.5 IU/mL, 33.3%; 0.05 IU/mL, 0%; respectively). Our results indicate the clinical sensitivity of CA V2.0, which is capable
of detecting > 50 IU/mL HCV RNA concentration in
clinical specimens.
Sensitivity and specificity tests
A total of 220 tests were performed with CA V2.0 for sensitivity and specificity. Among the sera from 100 CHC patients, HCV RNA was detectable in 99 (99%) samples with CA V2.0. HCV RNA was detectable in 1 (1.7%) of 60 CHB patients, and in 1 (1.7%) of 60 healthy blood donor samples. The sensitivity and specificity of CA V2.0 assay was 99% and 98.3%, respectively.
Clinical performance: comparison
between CA V2.0 and V1.0
Of the 84 naïve CHC patients receiving combination therapy with high dose conventional IFN (6 MU sub-cutaneously three times weekly) plus ribavirin, the SVR rate for HCV was 60.7%. At weeks 2, 4 and 8, HCV RNA was detectable with CA V1.0 in 43 (51.2%), 28 (33.3%), and 18 (21.4%) patients, respectively. On the other hand, HCV RNA was detectable with CA V2.0 in 51 (60.7%), 31 (36.9%), and 23 (27.4%) patients,
respectively (Table 1; p= 0.0047, 0.083, 0.025 at weeks
2, 4 and 8).
Table 1.Comparison of the sensitivity between CA V1.0 and CA V2.0 assays in HCV RNA detection amongst 84 naïve chronic hepatitis C patients receiving interferon plus ribavirin combination therapy*†
CA V1.0
Week 2 Week 4 Week 8
Positive Negative Positive Negative Positive Negative
CA V2.0
Positive 43 (51.2%) 8 (9.5%) 28 (33.3%) 3 (3.6%) 18 (21.4%) 5 (6.0%)
Negative 0 33 (39.3%) 0 53 (63.1%) 0 61 (72.6%)
p= 0.0047 p= 0.083 p= 0.025
*Comparison between CA V2.0 assay and CA V1.0 assay in sera of 84 naïve chronic hepatitis C patients receiving interferon (6 MU three times weekly) plus ribavirin for 24 weeks at weeks 2, 4 and 8 after initiation of combination therapy; †discrepancy between these two assays was analyzed with χ2for non-independent sample proportions (McNemar’s test). CA= COBAS AMPLICOR; HCV RNA = hepatitis C virus ribonucleic acid.
Overall, as shown in Table 2, the positive predic-tion values (PPV) for CA V2.0 and CA V1.0 were 90.91% (30/33) and 90.24% (37/41) at week 2, 83.02% (44/53) and 82.14% (46/56) at week 4, and 78.69% (48/61) and 72.73% (48/66) at week 8. The negative prediction values (NPV) for CA V2.0 and CA V1.0 were 58.82% (30/51) and 67.44% (29/43) at week 2, 77.42% (24/31) and 82.14% (23/28) at week 4, and 86.96% (20/23) and 83.33% (15/18) at week 8. There was no significant difference between CA V2.0 and V1.0 for the prediction of SVR in combination therapy.
D
ISCUSSIONDespite the improvement achieved by third-generation EIA, seroconversion of anti-HCV would not be de-tected until 7 to 8 weeks in approximately 30% of patients after exposure [3]. Supplemental tests such as commonly used recombinant immunoblot assay (RIBA) are usually indicated to make a confirmation. RIBA is standardized and reproducible, but its clini-cal application is limited due to being time consum-ing and relatively expensive. Besides, in clinical practice, there will be some patients with normal liver function during long-term follow-up who will still test positive for RIBA. In contrast, HCV RNA can usually be detected in the patient’s serum within 10–14 days after infection [3]. Therefore, in order to confirm the presence of active infection, it would be more efficient to use nucleic acid tests for circulating HCV RNA.
An ideal qualitative HCV RNA assay should be specific, accurate, reproducible, and standardized. Moreover, it should also be competent as a monitoring tool in diagnosis and treatment. To meet these practi-cal requirements, two commercially available assays, the second generation branched DNA (bDNA) V2.0 assay (Quantiplex HCV RNA; Bayer Diagnostics) and the COBAS AMPLICOR HCV Monitor test (COBAS V2.0; Roche Diagnostic Systems) are widely used. The bDNA assay is convenient and has excellent reproducibility, but has a low rate of sensitivity. The COBAS AMPLICOR HCV monitor test, using compet-itive RT-PCR, has been semi-automated and modified to amplify all genotypes equally [6,7]. Our results demonstrated that the CA V2.0 showed good reliability in the triplicate validation test with standard HCV RNA panels. We also showed that samples containing
T
able 2.
Comparison of the pr
edictions of sustained vir
ologic r
esponse (SVR) rate between CA
V1.0 and CA V2.0* † CA V1.0 CA V2.0 W eek 2 W eek 4 W eek 8 W eek 2 W eek 4 W eek 8 Negative Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative Positive SVR ‡ Positive 37 14 46 5 48 3 30 21 44 7 48 3 Negative 4 29 10 23 18 15 3 30 9 24 13 20 PPV §(%) 90.24 82.14 72.73 90.91 83.02 78.68 NPV ⎜⎜(%) 67.44 82.14 83.33 58.82 77.42 86.96 *Pr
ognostic values for combination therapy in 84 chr
onic hepatitis C patients wer
e analyzed in parallel with both CA
V2.0 and CA V1.0 at weeks 2, 4, and 8; †positive ( +) and negative ( −) pr
edictive values for CA
V1.0 and CA
V2.0 wer
e analyzed using McNemar
’s test;
‡SVR is defined as clearance of ser
um HCV RNA
at the end of tr
eatment and thr
oughout the 24-week
follow-up period;
§p
values for PPV between CA
V1.0 and CA
V2.0 wer
e 0.923 at week 2, 0.904 at week 4, and 0.435 at week 8;
⎜⎜p
values for NPV between CA
V1.0 and CA
V2.0 wer
e
0.389 at week 2, 0.653 at week 4, and 0.745 at week 8. CA
= COBAS AMPLICOR; PPV = positive pr ediction value; NPV = negative pr
ediction value; HCV RNA
=
hepatitis C vir
us
> 50 IU/mL of HCV RNA were positive (100%), and became negative below this level (0–33.3%). This ver-ified the limit of detection of the COBAS AMPLICOR HCV assay version 2.0 to be 50 IU/mL. The limit of detection of the COBAS AMPLICOR HCV assay ver-sion 1.0 is 1,000 copies/mL [8–10]. Since an interna-tional unit is equivalent to approximately 0.93–3.1 copies as measured in the COBAS AMPLICOR HCV formats [11], CA V2.0 is approximately 10-fold more sensitive than CA V1.0 [6].
The competitive RT-PCR assay is highly sensitive but is associated with the risk of contamination. In some previous studies [8–10], CA V1.0 assay showed high sensitivity (94–100%) and specificity (98.0%). In our study, the results among CHC, CHB, and healthy blood donors were compared in terms of serology and clinical diagnosis. CA V2.0 assay showed good sensitivity (99%), specificity (98.3%), and had good reliability. It therefore appears to be a suitable system for the monitoring and diagnosis of HCV infection. Only one patient (who had a clinical diagnosis of CHC but a negative result with CA V2.0) probably had (1) a low level of circulating HCV RNA below the analytic sensitivity of CA V2.0 and (2) a PCR priming site mutation.
Pretreatment HCV RNA measurements have been found to be one of the independent predictors of a sustained response after combination therapy with IFN and ribavirin [7,12]. Moreover, recent studies have shown that treatment strategies were mainly deter-mined or tailored by HCV RNA measurements before and/or during treatment [4,5]. The early disappear-ance of serum HCV RNA during antiviral therapy has been found to be associated with a favorable long-term response [13–17]. Our data showed that CA V2.0 had better performance than CA V1.0 regarding clinical performance as well as validation panel. It has been shown that CA V2.0 rather than V1.0 could
predict the outcome of IFN therapy in both serotype 1 and serotype 2 HCV [18]. However, CA V2.0 did not yield a higher predictive value of SVR to combina-tion therapy with standard IFN and ribavirin in our study. This might suggest that COBAS AMPLICOR HCV RNA assays, both CA V1.0 and CA V2.0 (com-bination or isolation), are an ideal method for quali-tative monitoring of treatment. Overall, as shown in Table 3, CA V2.0 offers several advantages over CA V1.0 [6,9,11]. Further studies in terms of quantitative measurements, especially during different stages of treatment, are needed to elucidate and compare their impacts on viral kinetics.
In conclusion, CA V2.0 showed good sensitivity and specificity and had good reliability in triplicate test. The lower limit of detection for CA V2.0 was 50 IU/mL. CA V2.0 is more sensitive than CA V1.0 in the detection of HCV RNA after the beginning of IFN plus ribavirin therapy for CHC patients. Both CA V2.0 and V1.0 showed that the viral load was an indi-cator of SVR. However, there is no significant differ-ence between CA V2.0 and V1.0 for predicting SVR in combination therapy.
R
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Table 3.Comparison between CA V1.0 and CA V2.0*
Assay Method Hands on time (hr) Overall workload time Limit of detection
COBAS AMPLICOR Manual RT-PCR ∼ 8 7 min/specimen 1,000 copies/mL
HCV 1.0 (CA V1.0)
COBAS AMPLICOR Automated RT-PCR ∼ 2 5 min/specimen 50 IU/mL
HCV 2.0 (CA V2.0)
*CA V2.0 offers some advantages over CA V1.0: CA V2.0 is approximately 10-fold more sensitive; CA V2.0 amplified all genotypes with similar efficiency, which improved sensitivity for non-1 genotypes HCV; by using the parallel-run feature of the instrument, the throughput can be increased to 3–4 runs per day.
5. Fried MW, Shiffman ML, Reddy KR, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 2002;347:975–82.
6. Doglio A, Laffont C, Caroli-Bosc FX, et al. Second gen-eration of the automated Cobas Amplicor HCV assay improves sensitivity of hepatitis C virus RNA detec-tion and yields results that are more clinically relevant.
J Clin Microbiol 1999;37:1567–9.
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10. Lunel F, Cresta P, Vitour D, et al. Comparative evalua-tion of hepatitis C virus RNA quantitaevalua-tion by branched DNA, NASBA, and monitor assays. Hepatology 1999;29: 528–35.
11. Lee SC, Antony A, Lee N, et al. Improved version 2.0 qualitative and quantitative AMPLICOR reverse transcription-PCR tests for hepatitis C virus RNA: cali-bration to international units, enhanced genotype reac-tivity, and performance characteristics. J Clin Microbiol 2000;38:4171–9.
12. Poynard T, Marcellin P, Lee SS, et al. Randomised trial of interferon alpha2b plus ribavirin for 48 weeks or for 24 weeks versus interferon alpha2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus. International Hepatitis Interventional Therapy Group (IHIT). Lancet 1998;352:1426–32.
13. Rossini A, Artini M, Levrero M, et al. Hepatitis C virus (HCV) RNA determination after two weeks of induc-tion interferon treatment is an accurate predictor of nonresponse: comparison of two treatment schedules.
Dig Dis Sci 2001;46:2389–95.
14. Toyoda H, Kumada T, Nakano S, et al. Significance of early measurement of serum hepatitis C virus RNA in predicting response to interferon therapy in patients with chronic hepatitis C. Eur J Gastroenterol Hepatol 1997;9:245–9.
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17. Kakumu S, Aiyama T, Okumura A, et al. Earlier loss of hepatitis C virus RNA in interferon therapy can predict a long-term response in chronic hepatitis C. J Gastroenterol
Hepatol 1997;12:468–72.
18. Kawai S, Yokosuka O, Imazeki F, et al. Evaluation of the clinical usefulness of COBAS AMPLICOR HCV MONITOR assay (ver2.0): Comparison with AMPLICOR HCV MONITOR assay (ver1.0) and HCV core protein level. J Med Virol 2002;68:343–51.
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